Hospital-acquired infections are a major cause of mortality and morbidity in intensive care units, and critically ill patients are particularly susceptible to bacterial infections due to immunosuppression. Since an increasingly greater portion of the hospital-acquired infections are caused by multidrug-resistant organisms, new therapeutic approaches are needed to prevent and treat bacterial infections in intensive care units. Our long- term goal is to develop an effective therapeutic strategy to overcome immunosuppression in critically ill patients through bolstering the bactericidal activity of neutrophils. Critically ill patients often display increased oxidative stress and have decreased levels of glutathione (GSH), a major intracellular antioxidant. GSH is generated through two pathways: de novo synthesis and regeneration from oxidized glutathione (GSSG). Recent studies in our laboratory demonstrated that mice lacking glutathione reductase (Gsr), which catalyzes GSH regeneration from GSSG, are highly vulnerable to infection by several bacterial species associated with hospital-acquired infections, as indicated by high rates of death, severe organ damage, and enhanced inflammation. The underlying cause of the increased vulnerability to bacterial infection is the failure to kill the infecting organism. In the absence of Gsr, neutrophils, the primary white blood cells responsible for bacterial eradication, cannot efficientl ingest and kill the infecting bacteria, likely due to excessive oxidative damage to the neutrophils themselves. Our studies indicate that GSH regeneration is pivotal for neutrophil-mediated host defense, raising the intriguing question of whether excessive oxidative stress is itself responsible for the immunosuppression seen in critically ill patients. Our findings strongly suggest that de novo GSH synthesis will be crucial for an effective immune defense against bacterial pathogens, since Gsr activity depends on de novo GSH synthesis, i.e. without GSH there can be no GSSG. The rate-limiting step in de novo GSH synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (Gclc) and a modulatory subunit (Gclm). The transcription factor Nrf2 regulates the expression of Gsr, Gclm, and Gclc, and thus Nrf2 modulates both de novo GSH synthesis and GSH regeneration from GSSG. The central hypothesis of our proposal is that the GSH antioxidant system modulated by Nrf2 facilitates host defense by maintaining bactericidal activity in neutrophils. The objectives of this proposal are to determine the role of GSH in neutrophil-mediated host defense and to assess the efficacy of Nrf2 activation as an approach to enhance host defense. The specific aims of this R21 proposal are to define the functions of the two GSH-generating pathways in anti-bacterial immune defense (Aim 1), and to evaluate the efficacy of Nrf2 activation as a therapeutic strategy to bolster neutrophil bactericidal activity (Aim 2). Our studies will elucidate a potential mechanism underlying immunosuppression in critically ill patients. Our studies will also facilitate the development of novel therapeutic drugs for the prevention and treatment of hospital-acquired infections in intensive care units.